Method for determining in vivo concentration of a metabolite

ABSTRACT

Method for determining in vivo concentration of a metabolite that includes administering a contrast agent to a subject, allowing the contrast agent to disperse to tissue of interest, performing CEDST MRI analysis of the subject, and comparing the results to known in vitro results to determine metabolite concentration.

PRIORITY CLAIM

This application is a divisional of U.S. patent application Ser. No.09/959,138, filed Oct. 17, 2001, (now U.S. Pat. No. 6,963,769 issuedNov. 8, 2005) which was the National Stage of International (PCT)Application No. PCT/US00/10878, filed Apr. 20, 2000, which claims thebenefit of U.S. Provisional Application No. 60/130,532, filed Apr. 21,1999, all of which are incorporated herein by reference.

FIELD

The present invention concerns a method for enhancing the quality (e.g.,contrast) of images produced by magnetic resonance imaging, particularlyimages produced in vivo, using chemical exchange dependent saturationtransfer and contrast agents.

BACKGROUND

Magnetic resonance imaging (MRI) provides a method for non-invasivelyobtaining diagnostic images of the body. MRI is now an indispensablediagnostic tool, and methods for improving the quality of the imageproduced are needed to facilitate image interpretation and provideadditional diagnostic information.

A. External Contrast Agents

Conventional MRI images of biological tissues reflect a combination ofspin-lattice (T1) and spin-spin (T2) water proton relaxation. Externallyadministered contrast agents, which enhance the relaxation rate of waterprotons, have been developed to enhance natural MRI contrast. Commonlyused external contrast agents include paramagnetic chelated metal ions,such as gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) andchelated metal Gd-DOTA. Casali et al., Acad. Radiol., 5:S214-8, (1998).The usefulness of these metal chelates as contrast agents for in vivoimaging is substantially limited by toxicity and T2 effects.

As an alternative to metal ions, other external MRI contrast agents,including iopamidol, arginine, serine and glycine, have been examinedfor their ability to enhance contrast in vitro. Aime et al., Invest.Radiol., 23:S267-70 (1988). These external contrast agents enhance MRIcontrast by decreasing the T2 signal, which is not very specific and canbe influenced by many factors.

B. Saturation Transfer

Previous studies showed that by saturating protons of small metabolites(i.e. ammonia) that can undergo chemical exchange with other materials,such as water, an associated decrease in the intensity of the waterproton signal resulted in a several-order magnitude increase insensitivity compared to direct detection. Wolff and Balaban, J. Magn.Reson., 86:164 (1990). This observation demonstrated that protonexchange can be imaged using saturation transfer methods in vitro.

Proton chemical exchange between water and metabolites is a commonprocess in biological tissues. Metabolite/water proton chemical exchangecan range from fast-to-intermediate-to-slow, depending on the chemistryof the exchange sites, temperature, pH and other factors. Strategieshave been presented to image the distribution of chemical exchange usingsaturation transfer (ST) in the magnetization preparation period of animaging sequence. Hsieh and Balaban, J. Mag. Res., 74: 574 (1987);McFarland et al., Mag. Reson. Imag., 6:507 (1988); Wolff and Balaban, J.Magn. Reson., 86:164 (1990). ST is most effective underslow-to-intermediate exchange conditions where the exchanging spins canbe adequately resolved and sufficient exchange occurs between themolecules, relative to T1, to detect transfer of the saturated protons.This limitation reduces the number of reactions that can be detectedwith ST; however, it may improve the specificity of the measurement incomplex biological tissues.

C. Intrinsic Tissue Contrast and ST

Saturation transfer methods known prior to the present invention relyprimarily on the patient's intrinsic macromolecules as the sole sourceof bound protons. The presence of ethanol also has been used to providefor an alternative source of bound protons. Govindaraju et al., Alcoholand Alcoholism, 32(6):671-681 (1997); Meyerfhoff et al. Alcoholism:Clinical and Experimental Research, 20(7):1283-1288 (1996). The effectobserved by these authors is due to dipolar interactions between waterprotons and free ethanol protons, not chemical exchange. Distinguishingbound protons from free protons in vivo is complex, making irradiationof solely bound protons difficult. In addition, although the intrinsicmacromolecules of some tissues readily undergo proton chemical exchange,other tissues do not. These factors have limited MRI contrastenhancement.

Nevertheless, intrinsic tissue contrast and saturation transfer havebeen used for imaging, For example, Balaban et al., U.S. Pat. No.5,050,609, which is incorporated herein by reference, describes usingsaturation transfer to enhance MRI contrast of tissues, polymers andgeological samples. Wolff and Balaban demonstrated exchange betweenirradiated bound protons with free protons using MRI saturation transfermethods in vivo. Wolff and Balaban Magn. Reson. Med., 10:135-144 (1989).The maximum amount of decrease observed in the free proton pool was 70%.This decrease was observed only in certain tissues, such as the rabbitkidney. Kajander et al. observed the greatest MRI contrast enhancementin striated muscle, but only modest enhancement in the liver, kidneycortex and spleen. Kajander et al., Magn. Reson. Imag., 4:413-7 (1996).Thomas used saturation transfer to improve the details of small vesselangiography to increase the contrast of breast and brain lesions, and toprovide greater details of the knee and cervical spine. Thomas, Radiol.Technol., 67:297-306 (1996).

SUMMARY

Intrinsic tissue contrast and saturation transfer has been used forimaging. Due to the amount of proton transfer in the kidney medulla,both the MRI signal and sensitivity increased, which enhanced the MRIcontrast of the kidney medulla. Guivel-Scharen et al., J. Magn. Reson.,133:36-45 (1998). However, no MRI contrast enhancement was observed whenthe intrinsic molecules of the liver and brain were used. Therefore,despite these previous methods, there still is a need for methods forenhancing the quality of images produced by MRI to facilitate imageinterpretation and provide additional diagnostic information.

The method of the present invention is useful for enhancing the contrastof MRI images, including images produced in vivo, using chemicalexchange dependent saturation transfer (CEDST). One feature of thepresent invention involved identifying contrast agents which containchemical groups having the appropriate proton exchange and chemicalshift properties at physiological pH and temperature to functioneffectively for performing CEDST MRI analyses in vivo. One embodiment ofthe method comprises administering at least one contrast agent to asubject (for example mammals, such as humans) in amounts effective toperform CEDST MRI analysis, and thereafter performing CEDST MRI analysisto produce an image of the subject.

There currently are two working embodiments for performing CEDST MRIanalysis. A first embodiment comprised: (a) selectively irradiating andsaturating an exchangeable proton or protons on an exogenouslyadministered molecule with an applied magnetic field; and (b)determining the effect of this saturation on the water proton MR signalamplitude. Transfer of saturated protons reduces the water proton signalamplitude. The distribution of this effect within a sample or subject isdetermined using conventional MRI imaging methods for determiningtopology of the water proton MR signal amplitude.

A second working embodiment for performing CEDST MRI analysis comprises:(a) selectively irradiating and saturating an exchangeable proton orprotons on an exogenously administered molecule with an applied magneticfield; (b) applying a selective irradiation with an equal but oppositeΔωCA from the water proton resonance position, thereby providing a firstimage set with the irradiation ±Δω_(CA); (c) producing a second imageset by either (1) subtracting or (2) dividing the images of the set(i.e., +Δω_(CA) and −Δω_(CA)) to minimize the effects of macromolecularinterference, T2, T1 and irradiation field in-homogeneity.

The contrast agent can be administered as a solid, as a dispersion orsolution, such as an aqueous composition, as a mixture of two or moreagents, etc. Intravenous (IV) delivery of a contrast agent or agentsdissolved or suspended in a physiologically acceptable carrier orcarriers is one method which can be used for administering contrastagents.

Examples of contrast agents suitable for administration as exogenouscontrast agents for performing CEDST MRI analyses in vivo can beselected from the group consisting of: sugars, includingoligosaccharides and polysaccharides, such as dextran; amino acids, suchas 5-hydroxy-tryptophan (which also includes an indole —NH having a pKaof about 1.7) and including oligomers of amino acids and proteins;nitrogen-containing heterocycles generally; indoles, purines andpyrimidines; nucleosides; imidazole and derivatives thereof, such as2-imidazolidone and 2-imidazoldinethione; imino acids, includingazetidines, such as azetidine-2-carboxylic acid, pyrolidines, such as4-trans-hydroxy-proline, and piperidines, such as pipecolinic acid;barbituric acid and analogs thereof, such as 2-thio-barbituric acid and5,5-diethylbarbituric acid; miscellaneous materials, such as guanidine,hydantoin, parabanic acid, and biologically active salts thereof; andmixtures of these contrast agents.

Suitable contrast agents often include at least one functional groupbearing a proton capable of chemical exchange. Examples of thesefunctional groups include, without limitation, amides, amines,carboxyls, hydroxyls, and sulfhydryls.

In addition to being useful for obtaining images by CEDST MRI havingsubstantially enhanced contrast compared to conventional MRI methods,the present method also is useful for determining certain conditions,such as pH and temperature, both in vitro and in vivo. One embodiment ofa method for determining pH comprised first determining, by CEDST MRI, aratio of (M_(O)−M_(S))^(Site 1)/(M_(O)−M_(S))^(Site 2) for a contrastagent having two exchangeable protons, and thereafter comparing the(M_(O)−M_(S))^(Site 1)/(M_(O)−M_(S))^(Site 2) ratios to a standard curveto determine pH of the desired tissue. A working embodiment of themethod used dihydrouracil as the contrast agent, which was provided asan aqueous composition having about 62.5 mM contrast agent. A standardpH curve was prepared by performing in vitro CEDST MRI analyses ofdihydrouracil as a function of pH. Ratios of(M_(O)−M_(S))^(Site 1)/(M_(O)−M_(S))^(Site 2) were then plotted togenerate the standard curve.

In yet another embodiment of the present invention, two or more contrastagents are used to determine pH both in vitro and in vivo. This methodprovided a greater dynamic range to the measurement. The methodcomprised first determining, by CEDST MRI, a ratio of M_(S)^(Site 2)(M_(O)−M _(S))^(Site 1)/[M_(S) ^(Site 1)(M_(O)−M_(S))^(Site 2)]for one or more contrast agents having two exchangeable protons, andthereafter comparing the M_(S) ^(Site 2)(M_(O)−M_(S))^(Site 1)/[M_(S)^(Site 1)(M_(O)−M_(S))^(Site 2)] ratios to a standard curve to determinepH of the desired tissue. Working embodiments of the method used eitherdihydrouracil or a combination solution of 5-Hydroxytryptophan and2-Imidazolidinethione as the contrast agent, which was provided as anaqueous composition having about 62.5 mM of each chemical in thesolution. Mixtures of other contrast agents may also be used to practicethe present invention. The contrast agents may be in the form ofpolymers. A standard pH curve is prepared by performing in vitro CEDSTMRI analyses of the contrast agent. Ratios of M_(S)^(Site 2)(M_(O)−M_(S))^(Site 1)/[M_(S) ^(Site 1)(M_(O)−M_(S))^(Site 2)]are plotted to generate the standard curve. To determining pH ofphysiological tissues in vivo, a standard curve is generated asdescribed, and the one or more contrast agents are administered to asubject, allowing the one or more contrast agents sufficient time tolocate in tissue of interest, determining in vivo by CEDST MRI analysisa ratio of M_(S) ^(Site 2)(M_(O)−M_(S))^(Site 1)/[M_(S)^(Site 1)(M_(O−M) _(S))^(Site 2)] using the one or more contrast agents,and comparing the M_(S) ^(Site 2)(M_(O)−M_(S))^(Site 1)/[M_(S)^(Site 1)(M_(O)−M_(S)) ^(Site 2)] ratio to a standard curve to determinepH of the tissue. Contrast agents which can be used include5,6-dihydrouracil, 5-hydroxy-tryptophan and 2-imidzaolidinethione,polymers thereof, and mixtures thereof.

Similar methods also can be used to determine other in vitro or in vivocharacteristics, such as the concentration of a specific metabolite insolution. The metabolite concentration can be determined using acontrast agent having exchangeable protons that are affected by thepresence of the metabolite. Like the pH determination described above,where protons on the contrast agent were affected by changes in the freeproton concentration, the (M_(O)−M_(S))^(Site 1)/(M_(O)−M_(S))^(Site 2)ratio can be used to determine the metabolite concentration. This ratiocan be determined in vivo by CEDST MRI analysis, and the determinedvalue compared to a standard curve to determine the concentration of themetabolite in the sample tested. A working embodiment of the method fordetermining phosphate concentration used dihydrouracil as the contrastagent, which was provided as an aqueous composition having about 62.5 mMcontrast agent at a fixed pH=6. A standard phosphate curve was preparedby performing in vitro CEDST analyses of dihydrouracil as a function ofphosphate concentration. Ratios of(M_(O)−M_(S))^(Site 1)/(M_(O)−M_(S))^(Site 2) were then plotted togenerate the standard phosphate curve. In a like manner, theconcentration of other metabolites, such as acetate and carbonate can bedetermined using the method of the present invention.

The present invention also provides a method for determiningphysiological temperature. One embodiment of the method for determiningphysiological temperature comprised first performing CEDST MRI analysisin vivo, and thereafter comparing the in vivo CEDST MRI results to astandard curve to determine physiological temperature. A workingembodiment of the method for determining temperature used barbituricacid as the contrast agent, which was provided as an aqueous compositionhaving about 62.5 mm contrast agent. However, other contrast agentswhich are temperature sensitive can also be used. A standard temperaturecurve was prepared by performing in vitro CEDST analyses of barbituricacid, at fixed pH and phosphate concentration, as a function oftemperature. The shape of the spectrum changes with changes intemperature. This shape can be characterized through a line-shapeanalysis of the entire ST spectrum, or of a subset of the spectrum, as afunction of temperature to derive the standard temperature calibrationcurve. A contrast agent possessing two proton chemical exchange sitescan also be used to determine temperature, like the pH and phosphatemeasurements, by the ratio of(M_(O)−M_(S))^(Site 1)/(M_(O)−M_(S))^(Site 2) as a function oftemperature.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription of several embodiments with reference to the accompanyingfigures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a CEDST spectra (M_(S) versus offset, in ppm) of 62.5 mM5,6-dihydro-uracil, containing 0-30 mM, pH 6 phosphate buffer,illustrating the effect of buffer concentration on the image produced bythe two exchangeable protons of 5,6-dihydrouracil at 2.67 ppm and 5.00ppm.

FIG. 2 is a CEDST spectra for 62.5 mM barbituric acid at 37° C. andvarying pH values.

FIG. 3 is a digital MR image of a 62.5 mM barbituric acid solution atdifferent pH values and NH₄Cl as a control, 37° C., illustrating theimage enhancement obtained using barbituric acid as a contrast agent.

FIG. 4 is a CEDST spectra of an aqueous 125 mM barbituric acid solution(pH=7.0, 37° C.) at two different saturation power levels, 8.8 and12.1×10⁻⁷T.

FIG. 5 is a graph of B1 power level×10⁻⁷ T versus M_(O)−M_(S) for theexchangeable protons resonating at 4.833 ppm and 5.000 ppm of an aqueous125 mM barbituric acid solution (pH=7.0, 37° C.).

FIG. 6 is a CEDST spectra of 62.5 mM 2-thio-barbituric acid in 20 mMphosphate buffer, 37° C., at pH values 4, 5, 6, 7 and 8.

FIG. 7 is a CEDST spectra of 62.5 mM 5,5-diethyl-barbituric acid, in 20mM phosphate buffer, 37° C., at pH values 4, 5, 6, 7, and 8.

FIG. 8 is a CEDST spectra of aqueous 5-hydroxy-tryptophan solutions 62.5mM, 37° C., 20 mM phosphate buffer at varying pH levels.

FIG. 9 is a digital control in vivo image of a rabbit bladder obtainedfollowing a saturating irradiation centered at −900 Hz (5.25 ppm/4T) offthe water resonance peak following administration of barbituric acid(125 mM/pH=7.4/20 mM phosphate buffer).

FIG. 10 is a digital in vivo image of a rabbit bladder obtainedfollowing a saturating irradiation centered at +900 Hz (5.25 ppm/4T) offthe water resonance peak following administration of barbituric acid(125 mM/pH=7.4/20 mM phosphate buffer).

FIG. 11 is a digital difference image (i.e., FIG. 9 image-FIG. 10 image)illustrating the location of the metabolite irradiated at +900 Hz (5.25ppm/4T) in the in vivo rabbit bladder following administration ofbarbituric acid (125 mM/pH=7.4/20 mM phosphate buffer).

FIG. 12 is a CEDST spectra showing of the in vivo rabbit bladderdescribed in FIGS. 8-10.

FIG. 13 is an ex vivo rabbit urine CEDST spectra, at 37° C. and a pH of5.2.

FIG. 14 is a CEDST spectra of a buffered barbituric acid solution (62.5mM, pH 5.0, 37° C., 20 mM phosphate buffer).

FIG. 15 provides the CEDST spectrum of 62.5 mM 5,6-dihydrouracil, T=37°C., at pH values of 5.0, 6.0 and 7.0.

FIG. 16 is M_(S) data versus pH for both exchangeable protons of5,6-dihydrouracil.

FIG. 17 is data ±SE from 3 experimental runs at each pH and curve fit ofratio (M_(O)−M_(S)), defined as(M_(O)−M_(S))^(Site 1)/(M_(O)−M_(S))^(Site 2) versus pH for5,6-Dihydrouracil (2.67 ppm: Site 1; 5.00 ppm: Site 2).

FIG. 18 provides the CEDST spectrum of a solution containing 62.5 mM5-hydroxytryptophan and 2-Imidazolidinethione at pH 7.0, 7.4, and 8.0.

FIG. 19 is M_(S) data versus pH for the exchangeable protons of5-hydroxytryptophan and 2-Imidazolidinethione.

FIG. 20 is data ±SE from 3 experimental runs at each pH and sigmoidalcurve fit of ratio M_(S) (M_(O)−M_(S)) versus pH for the combinationsolution (62.5 mM 5-hydroxytryptophan and 2-Imidazolidinethione, 5.33ppm: Site 1; 2.83 ppm: Site 2) where ratio M_(S)(M_(O)−M_(S)) is definedas M_(S) ^(Site 2)(M_(O)−M_(S))^(Site 1)/[M_(S)^(Site 1)(M_(O)−M_(S))^(Site 2)].

FIG. 21 is a CEDST spectra for: control solution containing 5-HT and2-IL, half the concentration of the control solution, and the controlsolution plus Gd-DPTA measured at pH=7.4, showing the effect of agentconcentration and water T1 on CEDST effects.

FIG. 22 is a CEDST spectra of 62.5 mM barbituric acid, (pH=7.4, in 20 mMphosphate buffer) measured at 7T at 25° C. and 37° C.

FIG. 23 is a CEDST spectra of 62.5 mM barbituric acid (pH 7.0, 20 mMphosphate buffer) measured at 7T for at 25° C. and 37° C.

FIG. 24 is a CEDST spectra of 62.5 mM barbituric acid (pH=6.5, 20 mMphosphate buffer) measured at 7T at 25° C. and 37° C.

FIG. 25 is a CEDST spectra illustrating the concentration dependence ofbarbituric acid at pH=7.0, temperature=37° C.

FIG. 26 is a CEDST spectra illustrating the concentration dependence ofbarbituric acid at pH=7.4, temperature=37° C.

DETAILED DESCRIPTION

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. It must be noted that as used hereinand in the appended claims, the singular forms “a” or ‘an’ or “the”include plural referents unless the context clearly dictates otherwise.Thus, for example, reference to “a contrast agent” includes a pluralityof such agents and includes reference to one or more agents andequivalents thereof known to those skilled in the art, and so forth.

I. Introduction

The present invention provides a method for obtaining MR images,particularly in vivo images, where the images provide better contrast,and therefore better quality, than prior known methods. Features of aworking embodiments of the invention include some or all of thefollowing: selecting one or more appropriate contrast agents;administering a contrast agent or agent, or composition comprising thecontrast agent or agents, to a subject; irradiating protons of thecontrast agent at a predetermined frequency (+Δω_(CA)) off the waterpeak, and thereafter providing an image; irradiating at a predeterminedfrequency (−Δω_(CA)) off the water peak, and providing a second image;and determining a third image provided by the subtraction or ratio ofthe first image relative to the second image. Contrast agents useful forpracticing the invention, and one embodiment of a method for obtainingin vivo MR images using such contrast agents, are described in moredetail below.

II. Definitions

The following terms are provided solely to aid in the understanding ofthis invention. These definitions should not be construed to have ascope less than would be understood by a person of ordinary skill in theart.

Amino Acid: An organic acid in which the carbon atoms in the hydrocarbonportion carry an amino group.

Chemical Exchange: A physical process whereby nuclides initially boundto a first compound become bound to a second compound, and hence arephysically transferred from the first to the second compound.

Chemical Exchange Dependent Saturation Transfer (CEDST): Refers to allsaturation transfer processes between molecules that are dependent onchemical exchange between the molecules.

Contrast Agent: A genus of materials having at least one proton that canchemically exchange for protons of another material, and which can beused to perform CEDST imaging.

Functional Group: A group of atoms, generally including a heteroatomsuch as oxygen, sulfur or nitrogen, bonded to one or more carbon atoms,to which an exchangeable proton also is attached (e.g., functionalgroups such as amines, hydroxyls or sulfhydryls) or which renders aproton attached to an adjacent atom more acidic (e.g., a proton bondedto a carbon atom a to a carbonyl carbon). Examples of functional groupsinclude, but are not limited to: amines (—R₃N, where R generally is analkyl group, an acyl group or hydrogen;, amides (—RCON—, where Rgenerally is an alkyl group or hydrogen); carbonyl groups [e.g., ketones(R₂C═O) and aldehydes (RHC═O)]; sulfhydryls (—SH); etc.

Magnetization Transfer (MT): Refers to through-space dipolarinteractions of nuclides within or between molecules.

MRI: Magnetic resonance imaging is a noninvasive diagnostic process thatuses an MR scanner to obtain images of objects, tissues, or bodies. AnMR scanner uses nuclear magnetic resonance to obtain images. The MRscanner includes (1) a body-encircling magnet that generates a strong,uniform magnetic field which interacts with radio waves to excite thenuclei of specific atoms, such as hydrogen, and (2) a detector thatdetects relaxation of the nuclei and transforms the detected signalsinto a visual image.

Mammals: Members of the Class Mammalia.

Pharmaceutically Acceptable Carriers: Includes all knownpharmaceutically acceptable carriers such as those described inRemington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 15th Edition (1975), herein incorporated by reference,which describes compositions and formulations suitable forpharmaceutical delivery of the contrast agents herein disclosed.Embodiments of the invention comprising one or more contrast agents canbe prepared with conventional pharmaceutically acceptable carriers,adjuvants and counterions as would be known to those of skill in theart.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise, in addition to the one or more contrast agents,injectable fluids that include pharmaceutically and physiologicallyacceptable fluids, including water, physiological saline, balanced saltsolutions, buffers, aqueous dextrose, glycerol, ethanol, sesame oil,combinations thereof, or the like as a vehicle. The medium also maycontain conventional pharmaceutical adjunct materials such as, forexample, pharmaceutically acceptable salts to adjust the osmoticpressure, buffers, preservatives and the like. The carrier andcomposition can be sterile, and the formulation suits the mode ofadministration.

For solid compositions (e.g., powder, pill, tablet, or capsule forms),conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, sodium saccharine,cellulose, magnesium carbonate, or magnesium stearate. In addition tobiologically-neutral carriers, pharmaceutical compositions to beadministered can contain minor amounts of auxiliary substances, such aswetting or emulsifying agents, preservatives, and pH buffering agentsand the like, for example sodium acetate or sorbitan monolaurate.

The composition can be a liquid solution, suspension, emulsion, tablet,pill, capsule, sustained release-formulation, or powder. The compositioncan be formulated as a suppository, with traditional binders andcarriers such as triglycerides.

Saturation Transfer (ST): “Saturation” refers to the destruction orrandomization of the net magnetization in a sample using an appliedmagnetic field with or without spatial magnetic field gradients.Transfer refers to a physical process whereby this saturation is passedbetween different molecules by through-space interactions or directchemical exchange from a first compound to a second compound.

Subject: Living multicellular vertebrate organisms, a category whichincludes human and veterinary subjects, for example mammals; farmanimals such as pigs, horses, and cows; laboratory animals such asrodents and rabbits; birds, and primates.

Δω_(CA): Refers to the chemical shift difference between the contrastagent proton chemical exchange site and the water proton resonancefrequency.

See, Balaban et al., U.S. Pat. No. 5,050,609, and Balaban et al.,“Detection of Proton Chemical Exchange Between Metabolites and Water inBiological Tissues,” J. Magn. Res.,” 133:36-45 (1998), which areincorporated herein by reference, for further information concerning MRIand ST.

III. Contrast Agents

One feature of the present invention is the identification/selection ofone or more appropriate contrast agents, that can be used to enhance thecontrast of an image of a material produced by MRI. The phrase “contrastagent” describes a genus of materials. Contrast agents typically havefunctional groups that provide (1) appropriate proton exchange, and (2)MRI chemical shift properties at physiological pH and temperature tofunction effectively for performing in vivo CEDST MR imaging.

A several thousand-fold enhancement of the proton signal can result viaa reduction in the water signal (M_(S)/M_(O)) based on Equation 1.M _(S) /M _(O)=[1/(1+k _(CA) T _(1W))]  Equation 1With reference to Equation 1, M_(S) is the magnitude of the water protonsignal in the presence of contrast agent proton saturation; M_(O) is themagnitude of the signal under control irradiation at the oppositefrequency offset; k_(CA) is the site proton exchange rate constant; andT_(1W) is the spin lattice relaxation rate of water protons.Guivel-Scharen et al., J. Magn. Res., 133:36 (1998). The site protonlifetime, τ_(CA), is the amount of time protons undergoing chemicalexchange remain bound to a particular molecule and is the inverse of thesite proton chemical exchange rate constant, k_(CA). Therefore,k _(CA)=1/τ_(CA)  Equation 2The amount of water affected per exchange site is similar to that inmetal-based T1 agents since both depend on the site proton lifetime,τ_(CA), and the diffusion rate of protons over the T1 time of water.

Factors that can be considered to select a contrast agent include: (1)the exchange rate, k_(CA), (the inverse of τ_(CA)), is in theslow-to-intermediate exchange rate domain, which is defined as:τ_(CA)Δω_(CA)>1  Equation 3where Δω_(CA) is the chemical shift difference (in radians/second)between the site and water, and τ_(CA) is the site proton lifetime[Dwek, Nuclear Magnetic Resonance (N.M.R.) in Biochemistry, Applicationsto Enzyme Systems, Oxford UK, (1973)]; (2) the Δω_(CA) is large enoughto support a large k_(CA) while satisfying the condition of Equation 3where Δω_(CA)>k_(CA); (3) a large Δω_(CA) also is desirable forspecificity since the B_(O) inhomogeneity can be >2 ppm; (4) highsolubility in aqueous, biologically acceptable carriers; (5) lowtoxicity; and (6) delivery of the contrast agent to apredetermined/selected tissue after administration.

Examples of classes of materials found to be useful in workingembodiments of the present invention for administration as exogenouscontrast agents include, but are not limited to, amino acids, sugars,nucleosides and their pyrimidine and purine bases, barbituric acid andanalogs thereof, nitrogen-containing heterocycles, includingheterocycles having plural exchangeable protons, such as two or more —NHgroups, and heterocycles having plural ring systems, imidazole andanalogs thereof, and imino acids and analogs thereof.

Examples of amino acids useful as exogenously administered MRI contrastagents include alanine, arginine, lysine, glutamine, tryptophan, and5-hydroxy-tryptophan. The amino acids used all were of the Lconfiguration, but amino acids having the D configuration also work topractice the method of the present invention. All stereoisomers ofcontrast agents discussed herein can be used to perform CEDST imaging.5-hydroxy-tryptophan has been used in working embodiments for in vivoimaging, as discussed in more detail below with reference to Table 1.

Monosaccharides, sugars, oligosaccharides (e.g., disaccharides such assucrose and lactose), polysaccharides, as well as the ketone andaldehyde analogs of such sugars, such as mannitol and sorbitol, areuseful for administration as contrast agents for practicing the methodof the present invention. Examples of sugars used to practice workingembodiments of the present invention include, without limitation,mannitol, mannose, sorbitol, sorbose, fructose, dextrose, galactose,sucrose, maltose and lactose. Structural formulas for certain of thesesugars and the disaccharide sucrose are provided below. Allhydroxylprotons (indicated in bold) of the sugars potentially areinvolved in the proton chemical exchange, and hence all hydroxylprotonsof the sugars are irradiated.

Examples of nucleosides and their pyrimidine and purine bases that areuseful as contrast agents for performing CEDST MRI include5,6-dihydrouracil, uridine and thymidine.

Barbituric acid and analogs thereof also are useful for exogenousadministration as contrast agents.

Suitable barbituric acid analogs typically have general structuralFormula 1.

With reference to Formula 1, R₁ and R₂ are independently selected fromthe group consisting of hydrogen and lower alkyl, where “lower” refersto hydrocarbon chains having 10 or fewer carbon atoms in the chain, andX is selected from the group consisting of oxygen and sulfur.2-thio-barbituric acid and barbital (5,5-diethylbarbituric acid) areexamples of barbituric acid analogs used to perform CEDST MRI accordingto the method of the present invention.

Imino acids also are useful as contrast agents for CEDST MRI. Iminoacids, such as (1) azetidines, e.g., azetidine-2-carboxylic acid,

(2) pyrrolidines, e.g., 4-trans-hydroxy-proline,

and (3) piperidines, e.g., pipecolinic acid,

have been used as contrast agents in working embodiments of the presentinvention.

The heterocyclic compounds, which includes indoles, generally, and the—NH site on the indole ring of 5-hydroxy-tryptophan, have both a maximal(M_(O)−M_(S)) in the physiological range and a desirable chemical shift(≧5 ppm), and thus remain in slow-to-intermediate exchange atphysiological pH. Both these features may be the result of theheterocyclic structure of these chemicals. The effect of theheterocyclic ring is to redistribute electrons within the ring, whichcan substantially affect the shielding of all nuclei attached to thering, and increase the ppm offset of those nuclei which are lessshielded.

Polymeric forms of contrast agents can provide better results for invivo administration as opposed to the monomeric precursors. One exampleis the polymerization of sugars into dextran. As seen in Table 1,dextran maintains the exchangeable site of the sugars despite beingpolymerized. One advantage of polymerizing contrast agents is thedelivery of more exchange sites per osmotically active particle comparedto the monomer. This may have important advantages in biologicalapplications where the osmolality of the contrast agent solution isimportant to reduce side effects.

Combinations of contrast agents also can be used, including combinationswithin a class of agents, such as a combination of sugars, andcombinations between two or more classes of contrast agents, such as anamino acid or a protein and a sugar or oligosaccharide. Examples ofcombinations used in a working embodiment of the present inventioncomprised combinations of thymidine (thymidine+pentose sugar) or uridine(uracil+pentose sugar) and phosphate, in addition to5-hydroxy-tryprophan and 2-imidazolidinethione.

Compositions comprising contrast agents, and combinations of contrastagents, also can be made to practice the method of the presentinvention. For example, contrast agents and pharmaceutically acceptablecarriers, materials for other diagnostic analyses, therapeutics, andcombinations of these materials, can be combined to provide acomposition useful for administration to a subject to practice themethod of the present invention.

Additional information concerning contrast agents, data collected usingsuch agents, and the methods used to obtain such data, is provided inthe Examples below and Table 1.

Contrast agents can be made to target a particular tissue. This can beaccomplished by, for example, conjugating a tumor-specific antibody orligand to a contrast agent(s) or polymers thereof, or by generatingpolymers that remain in the vasculature for angiography.

IV. Administering Contrast Agents

Once one or more appropriate contrast agents are selected, and suchagent or agents is administered to a subject. Known methods foradministering therapeutics and diagnostics can be used to administercontrast agents for practicing the present invention. For example,fluids that include pharmaceutically and physiologically acceptablefluids, including water, physiological saline, balanced salt solutions,buffers, aqueous dextrose, glycerol or the like as a vehicle, can beadministered by any method used by those skilled in the art. Methods ofintroduction include, but are not limited to, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,rectal, vaginal, and oral routes. The compositions may be administeredby any convenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, vaginal, rectal and intestinal mucosa, etc.) and may beadministered together with other biologically active agents.Administration can be systemic or local. In addition, the contrastagent(s) compositions may be introduced into the central nervous systemby any suitable route, including intraventricular and intrathecalinjection; intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir, such asan Ommaya reservoir. Solid compositions can be administered in the formof powders, pills, tablets, capsules, etc. The present invention alsoprovides pharmaceutical compositions which include contrast agents,alone or with a pharmaceutically acceptable carrier. In one example,homogeneous compositions of the one or more contrast agents includescompositions that are comprised of at least 90% of the contrast agentsin the composition.

Amounts of the one or more contrast agents sufficient to provide goodCEDST MRI results will be used, balanced by other considerations such aswhether the contrast agent used for a particular application mightproduce undesirable physiological results. The precise dose to beemployed in the formulation can also depend on the route ofadministration, and should be decided according to the judgment of thepractitioner and each subject's circumstances. In addition, in vitroassays (such as those disclosed herein in the following Examples) mayoptionally be employed to help identify optimal dosage ranges. Effectivedoses may be extrapolated from dose-response curves derived from invitro or animal model test systems. Examples of doses that can beadministered to a subject includes doses in the range of 0.0006-0.05moles of contrast agent(s)/kg of subject.

Contrast agents have been used successfully in concentrations rangingfrom about 0.2 mM (such as with dextran) to about 250 mM (such as withthe sugars). Thus, the amounts of the contrast agent or agentsadministered can range from moles, but more likely will be used inmillimolar-to-micromolar amounts. Polymerization of an agent, orcopolymers of various agents, significantly increases the number ofexchange sites while reducing the overall concentration of the agentitself.

Delivery Systems

Such carriers include, but are not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Thecarrier and composition can be sterile, and the formulation suits themode of administration. The composition can also contain minor amountsof wetting or emulsifying agents, or pH buffering agents. Thecomposition can be a liquid solution, suspension, emulsion, tablet,pill, capsule, sustained release formulation, or powder. The compositioncan be formulated as a suppository, with traditional binders andcarriers such as triglycerides. Oral formulations can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, and magnesiumcarbonate.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical contrast agent(s) compositions. Optionally associatedwith such container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.Instructions for use of the composition can also be included.

The pharmaceutical compositions or methods of treatment may beadministered in combination with other therapeutic treatments, such asother antineoplastic or antitumorigenic therapies.

V. Irradiating Protons of the Contrast Agent at a PredeterminedFrequency to Reduce the Intensity of the MRI Signal

Once an appropriate contrast agent or agents have been selected, theagents are administered to the subject and allowed to arrive at thetissue/region of interest. The subject is then analyzed by CEDST MRIanalysis to produce an image of the subject. There currently are twoworking embodiments for performing ST MRI analysis. A first embodimentcomprised: (a) selectively irradiating and saturating an exchangeableproton or protons on an exogenously administered agent with an appliedmagnetic field; and (b) determining the effect of this saturation on thewater proton MR signal amplitude. Transfer of saturated protons reducesthe water proton signal amplitude. The distribution of this effectwithin a sample or subject is determined using conventional MRI imagingmethods for determining topology of the water proton MR signalamplitude.

A second working embodiment for performing CEDST MRI analysis comprised:(a) selectively irradiating and saturating an exchangeable proton orprotons on an exogenously administered agent with an applied magneticfield; (b) applying a selective irradiation with an equal but oppositeΔω_(CA) from the water proton resonance position, thereby providing animage set with the irradiation ±Δω_(CA); (c) producing an image set byeither (1) subtracting or (2) dividing the images of the set (i.e.,+Δω_(CA) and −Δω_(CA)) to minimize the effects of macromolecularinterference, T2, T1 and irradiation field in-homogeneity. The finalimage has the greatest contrast (brightness) in regions containing theexternal contrast agent, as a result of decreasing the intensity of thewater peak.

The following examples are provided to illustrate certain features ofworking embodiments of the present invention. The scope of the inventionshould not be limited to those features exemplified.

EXAMPLE 1

This example describes the methods and results of in vitro studiesconducted to characterize the proton chemical exchange of severalcompounds with water. Candidate compounds (Table 1) were screened toidentify those containing proton chemical exchange sites with largeΔω_(CA), high solubility and appropriate chemical exchange rates atphysiological pH and temperature (pH 7.4 and 37° C.). Test compoundswere dissolved in HPLC grade water at concentrations denoted in Table 1with inorganic phosphate buffers to maintain pH. Gomori, MethodsEnzymol., 1:143, 1955. All chemicals were obtained from commercialsources (Aldrich Chemical Co., Milwaukee, Wis.; Mallinckrodt SpecialtyChemical Co., Paris, Ky.; and Sigma Chemical Co., St. Louis, Mo.).

Phosphate buffer concentration affected chemical exchange rates aspreviously described. Liepinsh and Otting, Magn. Reson. Med., 35:30,1995. FIG. 1 shows the CEDST spectra of 5,6-dihydro-uracil solutionscontaining varying phosphate buffer concentrations. Two specific protonchemical exchange sites are visible for 5,6-dihydro-uracil; one at 5.00ppm and one at 2.67 ppm. The site at 2.67 ppm was significantly affectedby changes in phosphate concentration; as the phosphate concentrationincreased, the M_(S) value increased, indicating that less exchange wasoccurring. The site at 5.00 ppm was substantially unaffected by changesin phosphate concentration. Therefore, the phosphate concentration washeld constant at 20 mM for the results reported in Table 1 to controlfor these effects.

TABLE 1 Compounds Tested as Potential Contrast Agents. Compound¹Conc(mM) FunctionalGroup ppm² pH³ $\frac{Ms}{Mo}$ Mo −Ms(%) Sugars⁴:Hydroxyl protons (—OH) Mannitol 250 mM —OH 1.000 7.0 0.89 9.0 Sorbitol250 mM —OH 1.000 7.0 0.88 7.3 Fructose 250 mM —OH 1.333 7.0 0.88 9.3Dextrose 250 mM —OH 1.500 7.0 0.89 8.7 Galactose 250 mM —OH 1.167 7.00.85 10.3 Sucrose 250 mM —OH 1.333 7.0 0.86 10.2 Maltose 250 mM —OH1.500 7.0 0.79 14.8 Lactose 250 mM —OH 1.333 7.0 0.68 20.9 Dextran5 1.75gm/100 ml 0.25 mM —OH 0.833 7.0 0.84 11.1 3.50 gm/100 ml 0.5 mM —OH1.167 7.0 0.91 8.1 7.00 gm/100 ml 1.0 mM —OH 1.167 7.0 0.81 13.6 14.0gm/100 ml 2.0 mM —OH 1.167 7.0 0.76 18.9 Amino protons Amino Acids^(6:)(—NH₂) L-Alanine 125 mM —NH₂ 3.000 4.0 0.36 67.4 L-Arginine 125 mM —NH₂3.000 4.0 0.36 65.8 L-Arginine 125 mM Guanidinium 2.000 5.0 0.33 57.7Protons L-Lysine 125 mM —NH₂ 3.000 4.0 0.34 66.2 L-Glutamine 125 mM —NH₂2.000 5.2 0.70 27.6 L-Tryptophan 35 mM —NH₂ 2.000 6.5 0.89 12.25-Hydroxy-Tryptophan⁷ 62.5 mM —NH₂ 2.833 4.0 0.57 41.6 Indole ring 5.3338.0 0.79 21.2 —NH Nucleosides and their pyrimidine Base protons andpurine bases^(7:) (—NH) 5,6 Dibydrouracil 62.5 mM 3-NH 5.000 6.0 0.7822.2 5,6 Dibydrouracil 62.5 mM 1-NH 2.667 7.0 0.77 22.2 Uridine⁸ 125 mM3-NH 6.333 4.0 0.65 34.7 Thymidine⁸ 125 mM 3-NH 6.333 5.0 0.65 34.8Pyrimidine ring Barbituric Acid and Protons its derivatives: (—NH)Barbituric Acid⁷ 62.5 mM —NH 5.000 6.5 0.68 32.5 2-Thio-Barbituric Acid⁷62.5 mM —NH 6.333 5.0 0.65 35.3 Barbital (5,5-Diethyl- 62.5 mM —NH 5.0004.0 0.82 14.2 Barbituric Acid)⁷ Imino Acids (their Azetidine, Baseprotons Pyrrolidine, and Piperidine forms) (—NH) Pipecolinic Acid⁶ 62.5mM —NH 3.33 5.0 0.81 19.3 4-Trans-Hydroxy-Proline⁶ 62.5 mM —NH 4.50 4.00.77 18.6 —NH 3.50 4.0 0.80 20.0 Azetidine-2-Carboxylic Acid 62.5 mM —NH3.50 5.0 0.74 25.5 Miscellaneous: Guanidine HCl⁴ 125 mM Guanidinium2.000 7.0 0.38 60.0 protons Hydantoin⁶ 62.5 mM —NH 5.667 4.0 0.81 18.7—NH 2.833 6.0 0.78 21.3 Parabanic Acid⁶ 62.5 mM —NH 5.167 7.0 0.79 20.5—NH 3.333 8.0 0.74 25.4 —NH 2.333 8.0 0.77 22.5 Imidazole and itsderivatives: 2-Imidazolidone⁶ Ring protons 1.167 5.0 0.68 30.4 (—NH)Ring protons 1.167 8.0 0.68 29.8 (—NH) 2-Imidazolidinethione⁶ Ringprotons 2.833 4.0 0.79 20.9 (—NH) Ring protons 2.833 7.0 0.65 34.5 (—NH)¹All compounds were evaluated at 37° C. and were dissolved in HPLC waterusing a 20 mM phosphate buffer unless otherwise noted. The power levelof the off-resonance saturation was 10.88 × 10⁻⁷T. ²ppm listed isrelative to the resonant frequency of water. ³The pH listed is where thegreatest proton chemical exchange effect was noted, save for thosesolutions only evaluated at pH = 7. ⁴These compounds were evaluated at asingle pH level of 7.0. ⁵Dextran molecular weigh: was approximately70,000 gm/mole; thus, concentrations listed (mM) for these solutions areapproximate. ⁶All solutions were evaluated at pH = 4, 5, 6, 7 and 8,except for L-Glutamine and L-Tryptophan. ⁷These solutions were evaluatedat pH = 4, 5, 6, 7 and 8. 5,6 Dihydrouracil, 5-Hydroxy-Tryptophan, andBarbituric acid solutions were also evaluated at pH = 6.5 and 7.4. ⁸Allsolutions of this compound were evaluated using a 2 mM phosphate buffer.Because phosphaw is the optimal physiologic catalyst of proton chemicalexchange (Liepinish and Otting, MRM 35: 30-42. 1996), the use of a 20 mMconcentration increased the exchange rate observed, increased thedesired exchange Ms/Mo ratio, and decreased the (Mo − Ms).With reference to Table 1, the compound, concentration of the compoundin 20 mM phosphate buffer, the protons and functional group bearing theprotons being irradiated are provided. Moreover, M_(S)/M_(O) andM_(O)−M_(S) are methods for expressing the difference between thecontrol and the experimental image, and hence the contrast, and isanalogous to the usage in radiology. The smallest M_(S)/M_(O) values,and conversely the largest (M_(O)−M_(S)) values, correspond to thegreatest contrast. An M_(S)/M_(O) value of less than 0.80, or an(M_(O)−M_(S)) value of greater than 20% currently is believed to be theminimal value to provide for good imaging contrast, although smallervalues may be useful for certain applications. An example of a ppm valuethat can be used for imaging is about 5 ppm or greater. For in vivoimaging an example of a pH value of the maximal (M_(O)−M_(S)) effect isfrom about 6.5 to 7.5, due to physiological conditions.

CEDST spectra were acquired at 7T using a Bruker AC-300 wide borespectrometer at 37° C. The observation frequency was set on the waterpeak and the decoupler was used to provide off-resonance saturation.Studies were conducted using a steady-state with irradiation (15seconds) over a range of irradiation frequencies±8.00 ppm from water.CEDST spectra were plotted in the form of water amplitude (M_(S)) versusirradiation frequency. Pulse sequence parameters: PW=8.0 μsec(1.47×10⁻⁶T; Flip angle=90°), one acquisition/Hz offset, 8192 datapoints, resolution of 0.97 Hz/pt, SW=8000 Hz.

Several classes of chemical exchange sites were evaluated (see Table 1).Sugar hydroxyl groups provided good chemical exchange sites at pH 7(M_(S)/M_(O) 0.89-0.68; 250 mM sugar). These compounds are not as usefulbecause their Δω_(CA) values (<2 ppm) are too small.

Sugar polymers, such as dextran, maintained the chemical exchange andshift properties observed with the monomeric sugars and also providednumerous exchange sites per osmole. Therefore, polymerization can beused to reduce the osmotic load.

Both the protons of the amino backbone of amino acids and theguanidinium R-group of arginine provide a good chemical exchange sitewith 2-3 ppm shifts (Table 1). However, these protons are not as usefulbecause they begin fast exchange by about pH 7.0. The indole ring —NHgroup on amino acid 5-hydroxy-tryptophan had useful properties. This isbecause it possessed a Δω_(CA) of 5.33 ppm and its exchange rates weresuitable over the pH range of 7.0-8.0.

Additional ring-NH groups including nucleosides, their pyrimidine andpurine bases, as well as derivatives of barbituric acid and imidazole,were evaluated (Table 1 and FIGS. 2-5). Several of these compoundsrevealed Δω_(CA) values in the desirable range (Δω_(CA)>3.0 ppm) andM_(S)/M_(O) values in the range of 0.7 for solutions at concentrationsof 62.5 mM.

The CEDST spectra of barbituric acid (62.5 mM, 37° C.) as a function ofpH are presented in FIG. 2, while an imaging series ofbarbiturate-containing phantoms is presented in FIG. 3. As shown in FIG.2, at 37° C. the exchange optimum for barbituric acid is approximatelypH 6.5. FIG. 2 also demonstrates that increasing the pH of thebarbituric acid caused the rate of proton exchange to approach fastexchange (where Δω_(CA)/K_(CA) is less than 1, or whereΔω_(CA)<<K_(CA)).

The results in FIG. 3 confirm those of FIG. 2. MR images of phantomswere collected on a custom-designed 4T system operating at roomtemperature (about 20° C.). The imaging sequence itself is a GRE(Gradient Recalled Echo) imaging sequence with an off-resonancesaturation pulse centered at an offset of 5.00 ppm (850 Hz for the fieldat 4 Tesla). The other parameters used were: 90 degree flip angle used,TE (Echo time)=9 msec, TR (Repetition time)=1.2 sec, 16 Sinc pulses,followed by crusher gradients (which completely dephased the transversemagnetization). Four test tubes are shown in FIG. 3, with eachcontaining a different solution. Three contained 62.5 mM barbituric acidat pH=7.4, 7.0, or 6.5. The fourth test tube contained 500 mM NH₄Cl atpH=5.0. These test tubes were placed inside the 4T NMR imaging system,irradiated at ±850 Hz, and both control and experimental images werecollected as described previously. FIG. 3 is the difference imagegenerated by subtracting the experimental image from the control image.All three solutions of barbituric acid were enhanced, while neither thesurrounding water nor the NH₄Cl control were significantly affected.Therefore, the signals in these phantoms were generated due to protonchemical exchange with barbituric acid. The brightest signal in thesephantoms (where the control and experimental image difference was thegreatest) was generated by barbituric acid at pH=7.4.

Concentration-dependent effects for barbituric acid were demonstrated(FIGS. 25-26). As shown in these figures, increases in agentconcentration increase the available contrast by increasing(M_(O)−M_(S)). Temperature (FIGS. 18-20) and saturation power effects(FIGS. 4-5) were also characterized. As shown in FIG. 4, increasing thepower level from 8.8×10⁻⁷ T to 12×10⁻⁷ T increases the contrast byincreasing (M_(O)−M_(S)). As shown in FIG. 5, increasing the saturationpower level increases the ST effect with an optimization at about16×10⁻⁷ T.

Two derivatives of barbituric acid, 2-thio-barbituric acid (FIG. 6)(barbituric acid with a sulfur substitution for the oxygen at C-2) and5,5-diethyl-barbituric acid (FIG. 7) (barbituric acid with two ethylgroups substituted for the hydrogens at C-5) also were examined atvarying pH levels. As shown in FIG. 6, the substitution of sulfur foroxygen at C-2 results in a peak shift from 5.00 ppm to 6.33 ppm, and ashift of the pH value at the peak ST effect, from pH=6.5 to pH=4.0. Thissubstitution improved the ppm offset value, but the change in pKaresulted in near fast exchange rates at physiological pH. Therefore,this compound is not as useful for use as a contrast agent forsaturation transfer at physiological pH. As shown in FIG. 7, the 5.00ppm site is completely lost due to the loss of both hydrogens at C-5 inbarbituric acid (compare to FIG. 2), and the pKa at the exchange sitedecreases from pH 12 to pH 7.

Barbituric acid is the parent chemical of all barbiturates, but is notitself pharmacologically active. Oral dosage toxicity is quite low,LD₅₀>5 g/kg. Goldenthal, Toxicol. Appl. Pharmacol., 18:185, 1971.Substitutions at groups of barbituric acid not involved in chemicalexchange are used to manufacture a wide variety of derivative drugs.This same site can be used to polymerize the compound, which wouldreduce the osmotic stress associated with mmoles of exchange sites ofmonomers.

The amino acid analog 5-hydroxy-tryptophan also was characterized forits proton chemical exchange with water. As shown in FIG. 8,5-hydroxy-tryptophan solutions combine an exchange site at 5.33 ppm withdecreasing M_(S) levels as the pH increased from pH 7.0 to pH 8.0,across the range of physiologic pH. Therefore, 5-hydroxy-tryptophanmaintains the desired slow proton chemical exchange at both a desirablepH range and ppm offset, making it a chemical useful in a workingembodiment of the present invention due to its combination ofcharacteristics.

EXAMPLE 2

This example describes the in vivo analysis of barbituric acid in arabbit bladder. A male New Zealand White rabbit (1.5 kg, HazeltonResearch Products, Denver Pa.) was initially anaesthetized with anintramuscular injection of a mixture of ketamine/acepromazaine (180 mg/2mg), intubated and placed on a Siemens 900c ventilator (Siemens MedicalSystems, Danvers Mass.). A catheter was placed in the marginal ear veinfor infusion of intravenous fluids to maintain volume status. Thiscatheter also was used to administer three doses of 50 mL barbituricacid solution (125 mM, pH=7.4, 20 mM phosphate buffer). Anesthesia wasmaintained with 2% isoflurane until sacrifice with 6 meq of KCl IV.

The barbituric acid reached the kidneys within 20 minutes and was thenimmediately filtered into the bladder. The rabbit appeared to bephysiologically unaltered (i.e. no change in blood pressure) in responseto the barbituric acid injection.

The rabbit was analyzed using a 4 Tesla magnet at the settings describedin Example 1. The images were obtained after irradiation at the ppmoffset characteristic of barbituric acid (5.25). FIG. 9 shows thecontrol image following saturating irradiation at −900 Hz (−5.25 ppm/4T)off the water resonance peak. The bladder and surrounding muscles arevisible. This control image is collected to correct for the broadbackground of saturation transfer from macromolecules (i.e. proteins,lipids) located underneath the water signal. Guivel-Scharen et al., J.Magn. Reson., 133:36-45 (1998), herein incorporated by reference. FIG.10 shows the image of the in vivo rabbit bladder following saturatingirradiation centered at +900 Hz (5.25 ppm/4T) off the water resonancepeak. This will irradiate the barbituric acid signal. As shown in FIG.10, the signal decreases in the bladder subsequent to irradiation at5.25 ppm. FIG. 11 shows the results of subtracting the in vivo image(FIG. 10) from the control image (FIG. 9). As shown in FIG. 11, the onlysignal remaining after subtracting the non-specific signal (FIG. 9), isin the bladder, not in the muscle or other tissues. These resultsdemonstrate that saturation transfer can be used to enhance the contrastproduced in an MR image of a tissue region containing an exogenouslyadministered contrast agent, such as barbituric acid.

Following sacrifice of the rabbit, urine was collected and evaluated onthe 7T system at 37° C. as described in Example 1. FIG. 12 shows the invivo CEDST spectrum of the bladder, obtained from the brightest sectionof FIG. 11. The peak at 5.25 ppm (900 Hz) off the water resonance(located at 0 ppm) demonstrates the specificity of the in vivo effect at5.25 ppm. FIG. 13 shows the ex vivo CEDST spectrum of the urinecollected from the sacrificed rabbit (pH 5.2, 37° C.). FIG. 14 shows theCEDST spectrum of a pure barbituric acid solution (62.5 mM, 20 mMphosphate buffer, pH 5.0, 37° C.). The spectrum shown in FIGS. 12-14demonstrate the validity of the specific location of the ppm offset usedin the in vivo imaging sequence (FIGS. 9-11). FIGS. 12-14 also confirmthe identity of the metabolite imaged during the in vivo analysis.

EXAMPLE 3

This example teaches how to determine pH using water proton NMR incombination with one or more exogenously added compounds. Magneticresonance spectroscopy can provide pH measurements using the chemicalshift differences between chemical moieties in vivo. Pan et al., Proc.Nail. Acad. Sci. USA., 85:7836; 1988; Mitsumori, J. Biochem.,97:1551;1985; Petersen et al., Magn. Reson. Med., 4:341;1987. However,most of these metabolites are in low concentration, making highresolution imaging or rapid determinations of pH difficult. Therefore, amethod was developed to obtain pH information from the intensity of thewater proton resonance to improve the signal-to-noise of pH measurementsby detecting pH-sensitive, water-proton chemical exchange with selectedmolecules using CEDST.

The pH affects the net chemical exchange reaction rate (K_(CA)) byvarying the concentration of the H⁺ and/or OH⁻ reactants. Thus, throughits effect on K_(CA), changes in pH will affect saturation transfersignals from water as indicated in Equation 1.M _(S) /M _(O)˜1/(1+k _(CA)T_(1W))  Equation 1As stated above, M_(S) is the magnitude of the water proton signal inthe presence of contrast agent proton saturation; M_(O) is the magnitudeof the signal under control irradiation at the opposite frequencyoffset; K_(CA) is the site proton exchange rate constant; and T_(1W) isthe spin lattice relaxation rate of water protons. To use CEDST todetermine K_(CA), the exchange site should have an adequate Δω_(CA) andbe in the slow-to-intermediate exchange domain (Equation 3), whereΔω_(CA)>K_(CA). After calibrating the effects of pH on K_(CA),M_(S)/M_(O) can be used to determine pH.

To resolve the exchange site chemical shift, Δω_(CA)/k_(CA) should begreater than 1, where Δω_(CA) is the chemical shift between waterprotons and the exchange site. A large Δω_(CA) permits a high exchangerate while remaining in the slow-to-intermediate exchange domain. A highexchange rate decreases the M_(S)/M_(O) ratio (see Equation 1) improvingthe CEST effect and subsequent signal to noise for the pH determination.A large Δω_(CA) also minimizes problems associated with magnetic fieldsusceptibilities and background macromolecular interference. Ward et al.J. Magn. Res., 143:79 (2000). The concentration of the exchange sitesmust be on the order of 40 mM to generate a significant M_(S)/M_(O)effects the limitations in proton Δω_(CA). The chemical shift of protonsin most biomolecules is quite small, but can be greatly enhanced withassociated metals (for example: Fe in myoglobin, Jue and Anderson. Magn.Reson. Med. 13:524 (1990)). The use of the chemical shift enhancingapproaches may extend the application of this method.

A method to eliminate obtaining additional measurements and backgroundeffects is to use a single molecule agent with two or more exchangesites with different chemical shifts and pH dependencies. Therelationship for each of the two sites on a dual-exchange site CEDSTagent (Site 1 and Site 2) can be represented as follows:(M _(O) −M _(S))^(Site 1) /M _(S) ^(Site 1) =k _(CA)^(Site 1)[Agent]^(Site 1) n ^(Site 1) T _(1W)  Equation 4(M _(O) −M _(S))^(Site 2) /M _(S) ^(Site 2) =k _(CA)^(Site 2)[Agent]^(Site 2) n ^(Site 2) T _(1W)  Equation 5The ratio of these two equations reduces to:M _(S) ^(Site 2)(M _(O) −M _(S))^(Site 1) /[M _(S) ^(Site 1)(M _(O) −M_(S))^(Site 2) ]=k _(CA) ^(Site 1)[Agent]^(Site 1) n ^(Site 1) /k _(CA)^(Site 2)[Agent]^(Site 2) n ^(Site 2)  Equation 6

As seen in Equation 6, the ratio of M_(S)^(Site 2)(M_(O)−M_(S))^(Site 1)/[M_(S) ^(Site 1)(M_(O)−M_(S) ^(Site)] isequivalent to the ratio of the site k₁ values (k₁ ^(Site)=k_(CA)^(Site)[Agent]^(Site)n^(Site)) that vary with pH. For a molecule withboth Site 1 and Site 2, the [Agent] and [n] terms cancel to leave aratio of the site K_(CA) values. If Site 1 and Site 2 are on differentmolecules, the ratio of (Agent) [n] is required. Based on therelationship illustrated in Equation 6 the M_(S)^(Site 2)(M_(O)−M_(S))^(Site 1)/[M_(S) ^(Site 1)(M_(O)−M_(S))^(Site 2)]data for two different sites plotted as a function of pH can be used tocreate a standard curve that eliminates effects associated with T_(1W),[Agent] and [n]. Based on these theoretical advantages of multipleexchange sites with different pH dependencies, this approach wasutilized.

Test compounds were dissolved in HPLC water and pH-specific inorganicphosphate buffers as described in Example 1 to maintain pH. Phosphatebuffer concentration affected CEDST results (see Example 1) and was heldconstant at 20 mM. Several chemical reagents, including5,6-dihydrouracil, 5-hydroxy-tryptophan, hydantoin, parabanic acid,sugars, amino acids, nucleosides, and imidazoles (Table 1), were testedfor their ability to undergo proton chemical exchange detected usingCEDST. Chemical shifts are reported relative to the water resonance. Thespectral dependence of CEDST was determined by sweeping the irradiationfrequency and while monitoring the water resonance. CEDST spectra wereacquired at 7T using a Bruker AC-300 wide bore spectrometer at 37°C.±0.1° C. The observation frequency was set on the water peak and thedecoupler was used to provide off-resonance saturation. Studies wereconducted using a steady-state with irradiation (15 seconds) over arange of irradiation frequencies±8.00 ppm from water. CEDST spectra wereplotted in the form of water amplitude (M_(S)) versus irradiationfrequency.

Saturation depends on the B1 power and the irradiation offset frequency.The appropriate power was determined by increasing power until nofurther decrease in M_(S) was observed. In most samples this occurredwith a B1˜14.7×10⁻⁷T. Due to the differences in T2 associated with theexchange rate, power requirements were calibrated for each experiment.Pulse sequence parameters: PW=8.0 μsec (1.47×10⁻⁶T; Flip angle=90°), oneacquisition/Hz offset, 8192 data points, resolution of 0.97 Hz/pt,SW=8000 Hz. Spin lattice relaxation time constants (T1) were obtainedwith inversion-recovery experiments. The range of inversion delays (Ti)was between 0.001 and 30 seconds, with a 30 second pre-delay.

The ratiometric analyses required separate M_(S) and M_(O)determinations for two sites. Three complete CEST spectra were analyzedto calculate the average and SEM of each of these parameters perexperimental solution. Plots of the ratios versus pH were then analyzedwith Sigma Plot to provide the Hill plot curve fit parameters and R2values

Initial studies identified several molecules with desirable chemicalexchange and shift characteristics. With a single site, the T_(1W) isdetermined, which reduces the speed and accuracy of the measurement. Toovercome this limitation, agents with two or more exchange sites withdifferent chemical shifts and pH dependencies can be used. In thisexample, agents with two different exchange sites were used in aratiometric fashion to determine the pH independent of T_(1W). Arepresentative molecule, 5,6-dihydrouracil, has two exchange sites, oneat 5.0 and the other at 2.67 ppm, each with a different pKa (FIG. 15).The exchange site at 5.0 ppm optimizes at pH 6, while the exchange siteat 2.67 optimizes at pH 7. At pH 5.0, the 2.67 ppm site is slow, whilethe 5.00 ppm site is faster resulting in a CEDST effect. At pH 6.0, the2.67 ppm site is now fast enough to be observed while the effect at 5.00ppm has increased further. At pH 7.0, the 2.67 ppm site has increasedwhile the 5.00 ppm peak has entered an intermediate exchange rateresulting in a chemical shift towards the water pool resonance and abroadening of the CEDST spectrum.

Plotting the individual M_(S) values as a function of pH (FIG. 16) showsthat each exchange site reaches a minimum M_(S) value as theintermediate exchange condition is reached. Above this point, the M_(S)value increases again due to exchange broadening and frequency shifteffects. A hyperbolic standard curve (FIG. 17) is produced by plottingthe ratio M_(S) ^(Site 2)(M_(O)−M_(S))^(Site 1)/[M_(S)^(Site 1)(M_(O)−M_(S))^(Site 2)] (±SE from 3 experiments; Site 1: 2.67ppm, Site 2: 5.00 ppm) as a function of pH. These results show that5,6-dihydrouracil is effective in the pH region of 6.5. However anotheragent with two exchange sties at 5.33 and 2.83 ppm, 5-hydroxy-tryptophan(5-HT), (see FIG. 8 and Example 1) is a good contrast agent atphysiologic pH. The pH dependence of the minimum M_(S) values occurredat pH 4 and 8 respectively, but did not significantly overlap inintermediate pH values. This resulted in a poor ratiometric performance.

Since both exchange sites on 5,6-dihyrouracil have the same dependenceon T_(1W) and the approach to fast exchange is independent of T1, therelationship between pH and ratio of (M_(O)−M_(S)) is independent ofT_(1W). The standard calibration curve is field dependent, however, dueto the field-specific effect on Δω_(CA).Δω_(CA)=(ppm)*(Field)(2π)  Equation 7

With reference to Equation 7, ppm is chemical shift difference ofcontrast agent in part-per-million units; and (Field) is the protonresonance frequency in MHz of the specific magnetic field (i.e., a 7Tfield operates at 300 MHz, a 4T field operates at 171 Mhz, and a 1.5Tfield operates at 63.6 MHz). The shift in Δω_(CA) when at a lowermagnetic field will change the slow-to-intermediate exchange limit(Equation 3) since the condition Δω_(CA)>K_(CA), must still besatisfied. The standard calibration curve is field dependent due to thefast exchange dependence on frequency difference.

Exchange sites on two different molecules can also be used to measurepH. This permits a larger number of sites to be evaluated as well as amethod of fine-tuning the pH sensitivity. These experiments wereconducted on solutions containing two separate chemical exchange agents.CEDST spectra from a solution containing 62.5 mM 5-Hydroxytryptophan(5-HT) and 2-Imidazolidinethione (2-IL) at different pH values is shownin FIG. 18. Plotting the individual M_(S) values as a function of pH(FIG. 19) shows that the 2-IL 2.83 ppm exchange site reaches minimumM_(S) at pH 7.0, while the 5-HT site at 5.33 ppm remains in slowexchange up to pH 8.0. The ratiometric analysis was performed on thesetwo sites at 5.33 and 2.83 ppm. Good sensitivity over the physiologicalpH range was observed (see FIG. 20).

The ability of the ratiometric technique to determine pH independent ofthe concentration of the contrast agent ([Agent]) or T_(1W) wasevaluated by altering [Agent] or adding Gd-DPTA (gadoliniumdiethylenetriaminepentaacetic acid). CEDST data from three solutions(all at pH 7.4) is presented in FIG. 21: Control (62.5 mM each 5-HT and2-IL) (solution 1); ½ Concentration of Control (31.25 mM each 5-HT and2-IL) (solution 2); and Control+Gd-DPTA (62.5 mM each 5-HT and 2-IL plus1 μl Gd-DPTA/ml) (solution 3). The T1 of the control solution (4.05+0.07sec, n=7), and solution 2 (T1=3.96+0.08 sec, n=7), were the same at ˜4sec, while solution 3 was reduced to 0.56+0.03 sec (n=7). CEDST datawere collected as a function of pH (pH 5.0, 6.0, 6.5, 7.0, 7.4, 7.7, and8.0) to perform the ratiometric analysis (Equation 6). As predicted(Equation 1), decreases in either T1 or [Agent] increased M_(S).However, the pH dependence of the ratio was unchanged at a given pH foreach solution using a paired t-test (solution 1 versus solution 2,p=0.22, d.o.f. =6; solution 1 versus solution 3, p=0.08, d.o.f.=6;solution 2 versus solution 3, p=0.63, d.o.f.=6).

The high concentration of exchange sites may provide additional protonbuffer capacity for the plasma or cytosol which could affect the pHmeasurement. However, the optimal pH for K_(CA) and M_(S)/M_(O) effectsdoes not necessarily correspond to the pKa of the molecule where thebuffering capacity is maximized. For example, the pKa of the 5.00 ppmsite on 5,6-dihydrouracil is ˜9.5 while pH 6.0 is the optimal pH forM_(S) effects, well away from its buffer capacity maximum. This occursbecause when the concentration of the base increases above a fewpercent, the exchange rate moves into the fast exchange limit in thismolecule. Thus, the proton buffering capacity of these probes may not bea problem for pH measurements.

This example demonstrates that detection of the chemical exchange rate,K_(CA), using CEDST techniques, can determine pH using the amplitude ofthe water signal and T_(1W). The use of the water proton signal indetecting pH resulted in a several hundred-fold enhancement ofsignal-to-noise over the direct chemical shift detection schemes. Thismay permit rapid kinetic studies or the eventual imaging of thedistribution of pH in biological samples. The multiple molecule approachallows the optimization of the chemical exchange sites for a given pHrange and may serve as a useful model system to guide the synthesis ofappropriate single molecule probes for in vitro and in vivo studies.

This method can be used to determine pH in vivo using external contrastagents and CEDST. The method would involve selecting one or moreappropriate contrast agents and preparing standard pH curves for the oneor more contrast agents as described above. The one or more contrastagents would be administered to a subject and CEDST MRI analysisperformed by irradiating the subject at the frequencies determined invitro for the contrast agent. The ratio M_(S)^(Site 2)(M_(O)−M_(S))^(Site 1)/[M_(S) ^(Site 1)(M_(O)−M_(S))^(Site 2)]would then be determined and compared to the standard curve to determinethe pH.

In vivo pH measurements are important diagnostic tools. For example, themethod can be used to determine the acidosis of a tumor. Thisinformation can then be used to select pharmaceuticals for tumortherapy, based on whether a tumor is going acidotic. As another example,in vivo pH measurements can be used to determine the pH of themyocardium to determine if it is ischemic (acidic pH). Furthermore, bycomparing the pH of the vasculature entering and leaving the kidney, onecan determine if the kidney is ischemic. The fact that these effects donot rely on the use of metals makes the ability to place these probesinside cells feasible using cleavable ester groups as used in opticaldyes.

EXAMPLE 4

This example describes the effects of temperature on CEDST MRI images.CEDST spectra of barbituric acid were obtained to evaluate thetemperature dependence of 62.5 mM solutions of barbituric acid at both25° C. and 37° C., and at pH values=7.0, 7.4, and 6.5. The ST spectrawere taken using the parameters described in Example 1.

FIG. 22 is a CEDST spectra illustrating the effects of temperature on a62.5 mM (pH=7.4, 20 mM phosphate buffer) barbituric acid solutionmeasured at 7T at 25° C. and 37° C. FIG. 23 is a CEDST spectraillustrating the effects of temperature on a 62.5 mM (pH=7.0, 20 mMphosphate buffer) barbituric acid solution measured at 7T at 25° C. and37° C. FIG. 24 is a CEDST spectra illustrating the effects oftemperature comparison on a 62.5 mM (pH=6.5, 20 mM phosphate buffer)barbituric acid solution measured at 7T at 25° C. and 37° C. FIGS. 22-24show that the CEDST spectra of contrast agents, in this case barbituricacid, can be affected by changes in temperature. In a manner similar tothat described in Example 3, temperature dependent standard curves canbe produced for a particular contrast agent. Standard temperaturedependent curves can be produced through in vitro CEDST analyses of theagent of interest, at fixed pH and phosphate concentrations, as afunction of temperature. The shape of a CEDST spectrum changes withchanges in temperature (see FIGS. 22-24). This shape can becharacterized through a line-shape analysis of the entire CEDSTspectrum, or of a subset of the spectrum as a function of temperature toderive the standard temperature calibration curve. A contrast agentpossessing two proton chemical exchange sites also can be analyzed aspreviously described, like the pH and phosphate concentrationmeasurements, by the ratio of(M_(O)−M_(S))^(Site 1)/(M_(O)−M_(S))^(Site 2) as a function oftemperature. Once standard curves are plotted, temperature of a sample,such as the in vivo temperature of tissue, can be determined by firstconducting CEDST MRI analyses of the sample using a known contrastagent, and thereafter comparing the results to the standard curve. Forexample, the method can be used to measure the temperature of tumor asit is being thermally-ablated.

EXAMPLE 5

This example concerns a method for determine metabolite concentrationsin vivo, such as a method for determining in vivo phosphateconcentration, using external contrast agents and CEDST. As describedabove in Example 1 and with reference to FIG. 1, the image generated bythe contrast agent 5,6-dihydrouracil depends on the phosphateconcentration. The ratio of the magnitude of the peaks at 5.00 ppm and2.67 ppm directly correlates with the phosphate concentration. Thus,phosphate concentration can be determined in vivo by (1) firstadministering a contrast agent, such as 5,6-dihydrouracil, to thesubject, (2) allowing the contrast agent to disperse to the tissue ofinterest, (3) performing CEDST MRI analysis of the subject byirradiating at ±2.67 ppm and ±5.00 ppm, (4) determining the ratio(M_(O)−M_(S))^(Site 1)/(M_(O)−M_(S)) ^(Site 2) (for this example, Site1:5.00 ppm, Site 2:2.67 ppm), for the subject, and (5) comparing theratio measured in vivo to known ratios determined in vitro to determinethe in vivo phosphate concentration. As the ratio of the peak magnitudeincreases, so does the phosphate concentration.

Having illustrated and described the principles of obtaining MRI imagesusing one or more contrast agents, it should be apparent to one skilledin the art that the invention can be modified in arrangement and detailwithout departing from such principles. In view of the many possibleembodiments to which the principles of our invention may be applied, itshould be recognized that the illustrated embodiments are only examplesof the invention and should not be taken as a limitation on the scope ofthe invention. Rather, the scope of the invention is in accord with thefollowing claims. We therefore claim as our invention all that comeswithin the scope and spirit of these claims.

1. A method for determining the in vivo concentration of a metabolite,comprising: administering a contrast agent to a subject; allowing thecontrast agent to disperse to tissue of interest; performing chemicalexchange dependent saturation transfer MRI analysis of the subjectcomparing results of the MRI analysis to known in vitro results todetermine metabolite concentration.
 2. The method according to claim 1where the contrast agent is 5,6-dihydrouracil.
 3. The method accordingto claim 2 where the metabolite is phosphate.
 4. The method according toclaim 2 and further comprising irradiating at 2.67 ppm and 5.00 ppm onboth sides of a water peak and then determining a ratio of(M_(O)−M_(S))^(Site 1)/(M_(O)−M_(S))^(Site 2), where site 1 resonates at5.00 ppm, and site 2 resonates at 2.67 ppm and M_(S) represents amagnitude of a water proton signal in presence of contrast agent protonsaturation and M_(O) represents a magnitude of a signal under controlirradiation at an opposite frequency offset.
 5. The method according toclaim 1 wherein the contrast agent is administered as an aqueouscomposition comprising an aqueous mixture of at least one contrast agentin amounts sufficient to perform chemical exchange dependent saturationtransfer MRI analysis, the contrast agent being selected from the groupconsisting of sugars and oligomers and polymers thereof, amino acids andoligomers and polymers thereof, nitrogen-containing heterocycles,nucleosides, imidazole and derivatives thereof, imino acids and analogsthereof, barbituric acid and analogs thereof, guanidine, hydantoin,parabanic acid, biologically active salts of the contrast agents, andmixtures of contrast agents.
 6. The method of claim 5 where thecomposition comprises from about 0.25 mM to 250 mM contrast agent. 7.The method of claim 1, where performing chemical exchange dependentsaturation transfer MRI analysis comprises: selectively irradiating andsaturating an exchangeable proton or protons of the contrast agent withan applied magnetic field; applying a selective irradiation with anequal but opposite chemical shift difference between the contrast agentproton exchange site and a water proton resonance frequency ()T_(CA)),thereby providing a first image set with the irradiation ±chemical shiftdifference between the contrast agent proton exchange site and the waterproton resonance frequency (±)T_(CA)); producing a second image setusing the first image set.
 8. The method of claim 1 where the contrastagent is 5-hydroxy-tryptophan.
 9. The method of claim 1 where thecontrast agent comprises a tryptophan.
 10. A method for determining invivo phosphate concentration, comprising: administering5,6-dihydrouracil to a subject; allowing the contrast agent to disperseto tissue of interest; performing chemical exchange dependent saturationtransfer MRI analysis of the subject by irradiating at 2.67 ppm and 5.00ppm on both sides of a water peak; determining a ratio of(M_(O)−M_(S))^(Site 1)/(M_(O)−M_(S))^(Site 2) where site 1 resonates at5.00 ppm and site 2 resonates at 2.67 ppm, where M_(S) represents amagnitude of a water proton signal in presence of contrast agent protonsaturation and M_(O) represents a magnitude of a signal under controlirradiation at an opposite frequency offset; and comparing thedetermined ratio to known in vitro ratios to determine phosphateconcentration.